CN114272378B - Use of a reagent for loss of CTTNBP2NL function in the preparation of medicines for treating diseases - Google Patents

Abstract

The invention provides a use of an agent which causes a loss of function of CTTNBP2NL in the manufacture of a medicament for the treatment of a disease. The use of an agent that causes a loss of CTTNBP2NL function in the manufacture of a medicament for the treatment of a disease caused by phosphorylation of STAT3 protein is mainly provided; the agent that causes the loss of function of CTTNBP2NL is an agent that inhibits expression of CTTNBP2NL or an agent that inhibits CTTNBP2NL from promoting STAT3 protein phosphorylation activity in tumor cells. The CTTNBP2NL deficiency reagent can be used for preparing medicines for treating tumors, autoimmune diseases and nerve diseases, and has good application prospects.

Description

A reagent for making CTTNBP2NL function deficient in the preparation of medicines for treating diseases the use of

technical field

The invention relates to the use of a reagent for making CTTNBP2NL function deficient in the preparation of medicines for treating diseases.

Background technique

Phosphorylation and dephosphorylation at specific sites on the protein surface are important post-translational modifications of proteins. This biological process plays an important role in intracellular signal transduction and changes in the activity state of enzymes. The most important difference between phosphorylation and dephosphorylation is that during phosphorylation, phosphate groups are added to amino acids of proteins by protein kinases whereas dephosphorylation is when phosphate groups are removed from Protein surface removal. Phosphate is usually derived from ATP or ADP. This process is ubiquitous in many physiological processes in organisms, especially in regulating protein function, localization, conformation, interaction and clearance. Phosphorylation and dephosphorylation are two key biological processes in living organisms. Protein phosphorylation and dephosphorylation are critical for signal transduction, cell division, protein translation, metabolism and survival. In cells, about 30% of proteins can be phosphorylated.

Furthermore, phosphorylation plays a key role in extracellular signaling. Neurotransmitters, hormones, cytokines, etc. produce effects by regulating the phosphorylation of target cells. More importantly, phosphorylation and dephosphorylation processes exist in almost all kinds of protein subtypes, such as structural proteins, enzymes, membrane channel proteins, signaling molecules, etc. There are more than 200,000 phosphorylation sites in human proteins. More than 500 different kinases are involved in the phosphorylation process. Protein phosphorylation also plays an important role in the development of cancer cells.

Cortical actin-binding protein 2 (CTTNBP2), a neuron-specific F-actin-associated protein, regulates cortical actin motility and controls dendritic spine formation by interacting with cortical actin. Sequence comparison indicated that CTTNBP2 N-terminal-like protein (CTTNBP2NL) is a homologous protein of CTTNBP2. Proteomic studies revealed that the striatum-protein phosphatase 2A (PP2A) protein complex is associated with CTTNBP2NL in HEK293 cells. CTTNBP2NL is associated with many physiological activities. However, it has not been seen that the CTTNBP2NL functional loss reagent of the present invention is used to prepare medicines and treat diseases.

Contents of the invention

In order to solve the above problems, the present invention provides a use of a reagent that makes CTTNBP2NL functionally deficient in the preparation of a drug for treating diseases caused by STAT3 protein phosphorylation; the reagent that makes CTTNBP2NL functionally deficient is a reagent that inhibits the expression of CTTNBP2NL or inhibits the expression of CTTNBP2NL Agents that promote phosphorylation activity of STAT3 protein in tumor cells.

Further, the drug is a drug for treating diseases caused by phosphorylation of Y705 site of STAT3 protein.

Further, the reagent for inhibiting the expression of CTTNBP2NL is a reagent for knocking out the CTTNBP2NL gene, knocking down the CTTNBP2NL gene or interfering with the expression of the CTTNBP2NL gene; preferably, the reagent is a reagent for RNAi, shRNA, or CRISPR/Cas9 methods;

Further preferably,

The RNA sequence of the reagent targeting CTTNBP2NL for the RNAi and shRNA methods is:

ATAACCCAGTTAGGTATTATA, GGTACTCACTAAGCGTTTATT, CTGAACTCCTGACACTATTTA, GCCCTCCATCCAGGGATTTAT, GCCACTCCTGCTTACTCATAT;

And/or, the RNA sequence targeting CTTNBP2NL of the reagent used in the CRISPR/Cas9 technology is:

ACTTTCATTGAAGAACGCTA, GCCGCTGCCTTTCTTCCTCA, TGTCACCTACATGCTAGAGA.

Further, the reagent that inhibits CTTNBP2NL from promoting the phosphorylation activity of STAT3 protein in tumor cells is a reagent that inhibits CTTNBP2NL from promoting the phosphorylation activity of STAT3 protein Y705 in tumor cells;

Preferably, the reagent is a reagent that mutates CTTNBP2NL; more preferably, the reagent is a reagent that phosphorylates the 488th and/or 527th positions of CTTNBP2NL, and it is further preferred that the 488th serine of CTTNBP2NL is mutated to glutamic acid and/or the 527th position A reagent for mutating serine at position 527 into glutamic acid;

Or, the reagent is a CTTNBP2NL mutant obtained by phosphorylation of the 488th and/or 527th positions of CTTNBP2NL, more preferably CTTNBP2NL 488th serine is mutated to glutamic acid and/or 527th serine is mutated to glutamic acid CTTNBP2NL mutants.

Further, the disease caused by phosphorylation of the STAT3 protein is tumor, autoimmune disease and/or neurological disease;

Preferably, the tumor is lung cancer, liver cancer, leukemia, colorectal cancer;

And/or, the autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, psoriasis;

And/or, the neurological disease is senile dementia, Parkinson's disease.

Further, the reagent for loss of CTTNBP2NL function is carried by adenovirus, adeno-associated virus, lentivirus or cells.

The present invention also provides a medicament for treating diseases caused by STAT3 protein phosphorylation, the medicament is an active ingredient of a reagent that causes CTTNBP2NL function loss; A reagent for STAT3 protein phosphorylation activity in tumor cells;

Preferably, the drug is a drug for treating diseases caused by phosphorylation of Y705 site of STAT3 protein.

Further, the reagent for inhibiting the expression of CTTNBP2NL is a reagent for knocking out the CTTNBP2NL gene, knocking down the CTTNBP2NL gene or interfering with the expression of the CTTNBP2NL gene; preferably, the reagent is a reagent for RNAi, shRNA, or CRISPR/Cas9 methods;

Further preferably,

The RNA sequence of the reagent targeting CTTNBP2NL for the RNAi and shRNA methods is:

ATAACCCAGTTAGGTATTATA, GGTACTCACTAAGCGTTTATT, CTGAACTCCTGACACTATTTA, GCCCTCCATCCAGGGATTTAT, GCCACTCCTGCTTACTCATAT;

And/or, the RNA sequence targeting CTTNBP2NL of the reagent used in the CRISPR/Cas9 technology is:

ACTTTCATTGAAGAACGCTA, GCCGCTGCCTTTCTTCCTCA, TGTCACCTACATGCTAGAGA.

Further, the reagent that inhibits CTTNBP2NL from promoting the phosphorylation activity of STAT3 protein in tumor cells is a reagent that inhibits CTTNBP2NL from promoting the phosphorylation activity of STAT3 protein Y705 in tumor cells;

Preferably, the reagent is a reagent that mutates CTTNBP2NL; more preferably, the reagent is a reagent that phosphorylates the 488th and/or 527th positions of CTTNBP2NL, and it is further preferred that the 488th serine of CTTNBP2NL is mutated to glutamic acid and/or the 527th position A reagent for mutating serine at position 527 into glutamic acid;

Or, the reagent is a CTTNBP2NL mutant obtained by phosphorylation of the 488th and/or 527th positions of CTTNBP2NL, more preferably CTTNBP2NL 488th serine is mutated to glutamic acid and/or 527th serine is mutated to glutamic acid CTTNBP2NL mutants.

Further, the disease caused by phosphorylation of the STAT3 protein is tumor, autoimmune disease and/or neurological disease;

Preferably, the tumor is lung cancer, liver cancer, leukemia, colorectal cancer;

And/or, the autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, psoriasis;

And/or, the neurological disease is senile dementia, Parkinson's disease.

Further, the reagent for loss of CTTNBP2NL function is carried by adenovirus, adeno-associated virus, lentivirus or cells.

RNA interference (RNAi) is a natural mechanism by which short interfering RNA (siRNA) induces gene silencing. Vector-based short hairpin RNA (shRNA) is an RNA interference (RNAi) technology used to study the function of unknown genes and can also be used for disease treatment. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) is an RNA-guided targeted genome editing tool that allows researchers to perform gene knockouts, insertions in cell lines and animals Foreign sequences, repair mutated bases and deleted sequences.

Overexpression of CTTNBP2NL can lead to increased phosphorylation of STAT3Y705, leading to disease. Losing the function of CTTNBP2NL, such as inhibiting the expression of CTTNBP2NL or inhibiting CTTNBP2NL to promote the phosphorylation activity of STAT3 protein in tumor cells, can treat diseases caused by phosphorylation of STAT3 protein, especially the disease caused by phosphorylation of Y705 site of STAT3 protein. The loss of CTTNBP2NL function can effectively reduce the phosphorylation of the Y705 site of STAT3 protein in tumor cells, significantly increase tumor cell apoptosis, inhibit tumor cell proliferation, and then inhibit tumor growth; at the same time, the loss of CTTNBP2NL function can reduce the production of autoantibodies, and the therapeutic system lupus erythematosus; it can also reduce the symptoms of rheumatoid arthritis, inflammatory bowel disease and psoriasis, and effectively treat rheumatoid arthritis, inflammatory bowel disease and psoriasis. Therefore, the reagent for loss of CTTNBP2NL function can be used as a drug for treating tumors, autoimmune diseases and neurological diseases, and has a good application prospect.

Apparently, according to the above content of the present invention, according to common technical knowledge and conventional means in this field, without departing from the above basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.

The above-mentioned content of the present invention will be further described in detail below through specific implementation in the form of examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Description of drawings

Figure 1 shows that the overexpression of CTTNBP2NL in Hela cells increases the phosphorylation of STAT3 Y705.

Figure 2 shows that the overexpression of CTTNBP2NL in HEK293 cells does not increase the phosphorylation of STAT3 Y705.

Figure 3 shows that the S488E and S527E mutations of CTTNBP2NL destroy its ability to promote STAT3 phosphorylation.

Figure 4 shows that the S488E and S527E mutations of CTTNBP2NL destroy its ability to promote STAT3 phosphorylation.

Figure 5 shows that knocking out CTTNBP2NL promotes cell apoptosis while reducing cell proliferation.

Figure 6 shows that interference with CTTNBP2NL expression reduces cell proliferation and promotes cell apoptosis.

Figure 7 shows that knocking out CTTNBP2NL inhibits tumor growth in nude mice subcutaneously.

Figure 8 shows that knocking down CTTNBP2NL inhibits the growth of subcutaneous tumors in nude mice.

Figure 9 shows that knocking down CTTNBP2NL alleviates the weight loss trend of SLE mice.

Figure 10 shows that knocking down CTTNBP2NL alleviates the production of autoantibodies in SLE mice.

Figure 11 shows that knocking down CTTNBP2NL alleviates the trend of increasing paw swelling thickness in mice with collagen-induced arthritis.

Figure 12 shows that knocking down CTTNBP2NL reduces the levels of inflammatory factors in inflammatory bowel disease.

Figure 13 shows that knocking down CTTNBP2NL reduces immune cell infiltration in skin lesions of psoriasis mice.

Figure 14 shows the effect of overexpression of CTTNBP2NL on the phosphorylation of STAT S568 in Hela cells.

Detailed ways

The raw materials and equipment used in the specific embodiment of the present invention are all known products, obtained by purchasing commercially available products.

Example 1. Overexpression of CTTNBP2NL causes increased phosphorylation of CTTNBP2NL in tumor cells

1. Experimental method

1. Cell culture

HEK293T, HEK293, HELA and other cells were purchased from ATCC and cultured in DMEM high-glucose medium, adding 10% fetal bovine serum and 1% penicillin and streptomycin. Cells were cultured in a 37°C cell incubator with a _CO2 concentration of 5%. When the cell growth density was close to 90%, the cells were passaged. Use the kit to identify mycoplasma once a month to ensure that the cells are free from mycoplasma infection. The preserved cells were cultured in 75cm disposable cell culture flasks, and the remaining cells were cultured in culture dishes of different specifications.

2. Carrier construction

(1) Extraction of cellular RNA

Use a 10cm cell culture dish to culture HEK293 cells or HELA cells. When the cell density is close to 70%, take out the culture dish, discard the cell culture medium, and use sterile PBS (after sterilization, add 1% double antibody, temporarily store at 4°C, 1 Weekly use, pH 7.2) After three quick washes, 1 ml TRZOL was added. Quickly hang down the cells with a cell spatula, transfer to a 1.5ml EP tube (RNase free) with a 1ml pipette gun (RNase free), and place on ice for 5min. Add 200 μl of chloroform, mix well, and place on ice for 5 minutes to observe the liquid layering. If the layering is good, proceed to the next step. Put the EP tube into a pre-cooled 4°C centrifuge, centrifuge at 12000g, 4°C for 10min. After centrifugation, take out the EP tube, gently suck 400μl of the colorless and transparent upper layer into a new 1.5ml EP tube (RNase free), add an equal volume of isopropanol (400μl) that has been pre-cooled in advance, mix it up and down, and then Place on ice for 10 min, then centrifuge at 12000 g for 30 min in a centrifuge at 4°C. After centrifugation, take out the EP tube and observe whether there is a white precipitate at the bottom of the EP tube. If there is a white precipitate, it is RNA. Gently aspirate the liquid in the tube, add 1ml of pre-cooled 75% ethanol to the EP tube, blow the precipitate with a pipette until the precipitate leaves the bottom of the EP tube. Then centrifuge in a centrifuge at 4°C, 12000g, for 5min. Aspirate all the supernatant (ethanol) as much as possible, open the cover of the EP tube in a fume hood, dry the residual alcohol, add 20 μl double distilled water (RNase free), and repeatedly pipette until completely dissolved. Take 2 μl into a new EP tube for concentration detection, and perform reverse transcription operation immediately after concentration detection.

(2) Determination of the concentration of DNA and RNA

The Nano Drop One instrument of Thermo Company was used for detection.

DNA concentration detection: After the power-on self-test, select the double-stranded DNA option. After the self-test is completed, add 1 μl of the same solvent as the sample to be tested to perform the background removal operation. After the background is removed, wipe off the residual liquid with absorbent paper and add 1 μl The test sample is tested, and the read value is the DNA concentration. At the same time, judge whether there is DNA contamination by observing the ratio between the absorption peaks of 260nm/280nm. The ratio between 1.7-1.9 is DNA, and the ratio greater than 1.9 is RNA.

RNA concentration detection: After power-on self-test, select the single-stranded RNA option, and the rest of the steps are the same as DNA detection.

(3) reverse transcription

Using the cDNA synthesis kit from Tiangen Company, take 1 μg RNA to a 200 μl PCR tube (RNase free), add 1 μl DNase and 2 μl Buffer, 7 μl ddH ₂ O (RNase free), 37°C for 20 min, to remove possible genomic DNA contamination. After removing the genomic DNA, add 2μl reverse transcriptase, 4μl 5X reverse transcription Buffer, 4μl ddH ₂ O (RNase free), put into PCR machine, reverse transcription program: 42℃, 30min→95℃, 5min→4℃ , 5min.

(4) PCR amplification of CTTNBP2NL mRNA full length

Use SnapGene software to design nested PCR primers outside the CDS region of CTTNBP2NL. The specific primer information is as follows:

Nested PCR Outer Primers:

Upstream: 5'-GAGGACTGAAGTGTGACTCTGCCG-3'

Downstream: 5'-GGACGAAATCTGATGGAATGTCAAAC-3'

Nested PCR internal primers:

Upstream: 5'-ATGAATCTGGAAAAACTCAGCAAGC-3'

Downstream: 5'-CTAGCTGCTGGTAGGCAAAAGTAACTC-3'

PCR system: 25 μl, using Tiangen Pfu enzyme mix (2X)

Template DNA: 1 μl;

Upstream and downstream primers: 1 μl each;

_ddH2O : 9.5 μl;

2X Pfu Mix: 12.5 μl.

PCR program: 95°C, 5min→95°C, 30s→55°C, 30s; 72°C, 3min→72°C, 10min→4°C, 5min. Number of cycles: 30. Because the T vector needs to be connected later, it is necessary to add 5 μl of ordinary Tag mix after the PCR, 95°C, 30s→60°C, 30s; 72°C, 5min, 4°C, 5min, and temporarily store the PCR product at -20°C in the refrigerator.

Nucleotide sequence:

SEQ ID NO.1: GAGGACTGAAGTGTGACTCTGCCG

SEQ ID NO.2: GGACGAAATCTGATGGAATGTCAAAC

SEQ ID NO.3: ATGAATCTGGAAAAACTCAGCAAGC

SEQ ID NO.4: CTAGCTGCTGGTAGGCAAAAGTAACTC

(5) Nucleic acid electrophoresis

Prepare 1% nucleic acid separation gel with 1X TAE Buffer: Weigh 4g of agarose and pour it into a 200ml Erlenmeyer flask. Measure 40ml of 1X TAE Buffer with a graduated cylinder, add it to the Erlenmeyer flask, shake well, heat to boiling in a microwave oven, keep at high temperature for 5 minutes, and take out the Erlenmeyer flask after the agarose is completely dissolved. Add 2μl GelRed, mix well, pour into gel plate, and insert 10-hole comb. Place at room temperature for 30 minutes. After the gel is solidified, take out the nucleic acid gel and put it into a horizontal electrophoresis tank. After loading the PCR product, 120v, 30min. After electrophoresis, transfer the nucleic acid gel to a gel imager, cut out the corresponding nucleic acid bands according to the size of the marker, put them into a 2ml EP tube, and carry out subsequent operations.

(6) Glue recovery

Use Tiangen Gel Recovery Kit. Add 300μl equilibration buffer to the collection column in advance, centrifuge at 7000g for 5min at room temperature. Take out the 2ml EP tube containing the DNA gel, crush it with a 1.5ml pipette tip, weigh the gel block with an electronic balance, add an equal amount of buffer PN (1ml PN to 1g gel), and dissolve on a hot block at 60°C 30min, during the period, mix up and down once to ensure that the nucleic acid gel is completely dissolved. Remove the EP tube and cool to room temperature. Transfer the dissolved liquid to a DNA collection column, centrifuge at 7000g for 5min at room temperature. After discarding the waste liquid, add 500 μl rinse solution (PW) to the collection column, centrifuge at 7000 g for 5 min at room temperature. After rinsing twice, the empty tube was centrifuged at room temperature for 5 min, and the residual liquid in the collection tube was dried in a fume hood, then 30 μl of eluent (TB) was added to the collection tube, incubated at 60°C for 10 min, 7000 g, and centrifuged at room temperature for 5 min. After the DNA concentration was determined by Nano Drop, follow-up operations were performed.

(7) Connection with T vector and transformation of competent cells

Use Quanshijin company T carrier, take 1 μg gel to recover PCR product, add 1 μl T carrier, adjust the volume to 5 μl with double distilled water, put it in a PCR instrument, and react at 37°C for 30 minutes to obtain the ligation product of CTTNBP2NL and T carrier, 4 ℃ Temporary storage. Competent cells were purchased from Trans T1. During transformation, the competent cells stored at -80°C were taken out and thawed on ice. Take 2.5 μl of the ligation product of CTTNBP2NL and T carrier, add it to 300 μl of competent cells, incubate on ice for 20 minutes, heat shock in a water bath at 42°C for 90 seconds, put it on ice immediately, add 200 μl of non-resistant liquid LB, Put it into a horizontal bacterial shaker, 37°C, 100 rpm, and recover for 30 minutes. Take out the recovered competent cells, put them in a centrifuge, centrifuge at 5000g, room temperature for 5min, suck off the supernatant in an ultra-clean bench, leave 100μl of bacterial solution, resuspend the bacterial pellet with a pipette gun, and take out the bacteria containing 100μg/ml For ampicillin-based solid LB bacterial culture plates, use glass rods to evenly spread the bacterial liquid on the surface of the bacterial culture plates. After upside-down cultivation in a bacterial incubator at 37°C for 12 hours, the growth state of the colony was observed.

(8) Bacterial monoclonal expansion culture and strain preservation

Take a 10ml bacterial culture tube, add 5ml liquid LB medium, and add 1X antibiotics. Pick a single colony with the tip of a 10 μl sterile pipette, drop the tip into a 10ml bacterial culture tube, and incubate in a horizontal bacterial shaker at 37°C and 200 rpm for 12 hours. Select the bacterial solution in the logarithmic growth phase, absorb 700 μl of the bacterial solution in an ultra-clean bench, add it to 300 μl of 50% glycerol (glycerin is sterilized in advance), mix it upside down, and store it in a -80°C refrigerator.

(9) Plasmid extraction

Plasmids were extracted using Quanshijin Plasmid Miniprep Kit. After two times of centrifugation, 2ml each time, a total of 4ml of bacterial liquid was collected into a 2ml EP tube, the supernatant was discarded, and 250μl of solution 1 (25mM Tris-HCl, 10mM EDTA, 50mM glucose, adjusted to pH 8.0) was added, vortexed After spinning and resuspending, add 250 μl of solution 2 (0.2N NaOH, 1% SDS), invert and mix well, let stand at room temperature for 5 min, add 350 μl of solution 3 (2M acetic acid, 3M potassium acetate, 75% alcohol), invert and mix well, Place at room temperature for 10 minutes, then centrifuge at 12000g for 10 minutes at room temperature. Transfer the supernatant to a collection column, centrifuge at 12000g for 2min at room temperature. Pour off the waste liquid in the collection tube, add 500 μl of washing solution to the collection column, centrifuge at 12000 g for 2 min at room temperature. Pour off the waste liquid in the collection tube, replace the collection tube with a new 1.5ml EP tube, add 30μl TB buffer to the collection column, incubate at 60°C for 10min, centrifuge at 12000g for 2min at room temperature, the liquid in the EP tube is the plasmid.

(10) PLVX-CTTNBP2NL-3x Flag plasmid construction (other plasmid construction principles and steps are basically the same)

The PLVX-3X Flag vector was used as the eukaryotic expression vector, and the vector was purchased from Addgene. Choose the double enzyme digestion scheme for construction. Upstream restriction site selection: XhoI, downstream restriction site selection: BamHI. Use NEB company endonuclease for double enzyme digestion reaction, reaction system: 50 μl, specific composition ratio:

DNA: 1 μg;

Cutsmart buffer: 5μl;

XhoI: 1 μl;

BamHI: 1 μl;

H ₂ O: Make up to 50 μl.

Digestion conditions: 37°C, 1h.

After enzyme digestion, add 5 μl 10X loading Dye for electrophoresis and gel recovery, and the gel recovery product is temporarily stored in a -20°C refrigerator. Take out the T carrier bacterial liquid containing CTTNBP2NL CDS, and directly perform bacterial liquid PCR. The PCR primers used contain XhoI and BamHI restriction sites and ATGC four bases as protective bases on both sides. After PCR, the PCR product is recovered by gelling. After double enzyme digestion and gel recovery, the product was temporarily stored in a -20°C refrigerator.

Ligation: The CTTNBP2NL CDS region fragment and the carrier fragment were ligated at a molar ratio of 3:1, using the T4 ligation kit of Quanshijin Company, ligation system: 20 μl, ligation conditions: 16°C, overnight ligation. The ligation product was transformed into competent cells, single clones were picked, and after the correct insertion fragment was confirmed by sequencing, a large number of plasmids were extracted.

(11) PLVX-CTTNBP2NL-mCherry plasmid construction (other plasmid construction principles and steps are basically the same)

The PLVX-C1-mCherry vector was used as the eukaryotic expression vector, and the vector was purchased from Addgene. Choose the double enzyme digestion scheme for construction. Upstream restriction site selection: XhoI, downstream restriction site selection: EcoRI. Use NEB company endonuclease for double digestion reaction. The remaining steps are the same as the construction process of PLVX-CTTNBP2NL-3x Flag plasmid.

(12) PLVX-CTTNBP2NL-AcGFP plasmid construction (other plasmid construction principles and steps are basically the same)

Using the PLVX-C1-mCherry vector as the initial vector, mCherry was replaced by AcGFP by homologous recombination. After the sequencing was correct, the PLVX-C1-AcGFP vector was used as the eukaryotic expression vector, and the double enzyme digestion scheme was selected for construction. Upstream restriction site selection: XhoI, downstream restriction site selection: EcoRI. Use NEB company endonuclease for double digestion reaction. The remaining steps are the same as the construction process of PLVX-CTTNBP2NL-3x Flag plasmid.

(13) PLVX-CTTNBP2NLC1-3XHA plasmid construction

Using the PLVX-3XFlag vector as the initial vector, the 3xFlag was replaced with 3xHA by homologous recombination. After the sequencing was correct, the PLVX-3XHA vector was used as the eukaryotic expression vector to select the double enzyme digestion scheme for construction. Upstream restriction site selection: EcoRI, downstream restriction site selection: BamHI. Use NEB company endonuclease for double digestion reaction. The remaining steps are the same as the CTTNBP2NL-3x Flag plasmid construction process.

3. Lentiviral packaging

The constructed viral backbone vector together with psPAX2 and pMD2.G packaging plasmids were transfected into 293T cells, PLVX:psPAX2:pMD2.G=1:2:1 (molar ratio). The cell supernatant was collected 48 hours after transfection, and the second cell supernatant was collected 72 hours after transfection. After concentrating the virus with PEG, aliquot it and temporarily store it in a -80°C refrigerator.

4. Construction of stable knockout monoclonal cell lines

293 cells were cultured in a 10cm cell culture dish and infected with CTTNBP2NL knockout lentivirus. After a period of infection, Puromycin was added to screen, and then the adherent cells were digested. After making a single cell suspension with DMEM medium containing penicillin and streptomycin, the Single cells are screened by flow cytometry. The obtained single cells continued to be cultured, and the growth status of the cells was observed under a microscope, and 20 monoclonal cells were selected for expansion culture, and the expression status of CTTNBP2NL was identified by Western-Blot method and gene sequencing method, and the CTTNBP2NL protein completely disappeared and the CTTNBP2NL CDS sequence was selected. Coded cells were used as a stable knockout monoclonal cell line.

5. Western blot

Use Co-IP and Western cell lysate from Beyontien Company. Specific steps: Take out the cultured cells or isolated mitochondria, wash the cell supernatant with PBS, add 100-400 μl of lysate according to the number of cells, and place on ice After 5 minutes, centrifuge at 12,000g at 4°C for 10 minutes, transfer the supernatant to a new EP tube, take out 5 μl for protein concentration determination, add 5X SDS protein sample buffer to the rest of the protein, place it on a constant temperature metal heat block, and denature at 95°C 5min, temporarily stored at -20°C.

Use the combination of 10% SDS separating gel + 5% SDS stacking gel for electrophoresis, electrophoresis conditions: 70V in the stacking gel, leave the stacking gel for about 20 minutes, and after entering the separating gel, adjust the voltage to 110V, and the separation time is about 60 minutes.

The membrane was transferred using SDS-methanol transfer buffer. Transfer conditions: 250mA, constant flow, transfer on ice for 90min, take out the PVDF membrane, block in 5% skimmed milk powder at room temperature for 30min, dilute the primary antibody with TBST, dilution ratio: 1:1000, 4°C, incubate overnight After the primary antibody was washed with TBST, the secondary antibody was added on a horizontal shaker at a dilution ratio of 1:2000, and incubated at 37°C for 1 hour. After washing the secondary antibody on a horizontal shaker with TBST, it was exposed on a chemiluminescence instrument and photographed.

2. Experimental results

By constructing CTTNBP2NL overexpression lentivirus and infecting HEK293 cells and Hela cells, a cell line stably expressing CTTNBP2NL was obtained through monoclonal screening. The changes of STAT3 phosphorylation in these two cell lines were detected by Western blot experiments. Overexpression of CTTNBP2NL in tumor cells significantly increased the phosphorylation of Y705 site of STAT3 protein in cells (Figure 1).

However, CTTNBP2NL fused with different tags were overexpressed in normal cells, including CTTNBP2NL-3xHA, CTTNBP2NL-3x Flag, CTTNBP2NL-GFP, and CTTNBP2NL-RFP. In HEK293 control cells and CTTNBP2NL overexpression cells, there was no phosphorylation phenotype at STAT3Y705 (Figure 2). The above results indicate that the overexpression of CTTNBP2NL can cause increased phosphorylation of STAT3Y705, and this phenomenon only exists in tumor cells, and normal cells are not affected.

Through base mutation, the serine (S) at position 527 was mutated to glutamic acid (E), and the serine (S) at position 488 was mutated to glutamic acid (E), two mutants were obtained, through Hela cells After overexpressing the above two mutants, it was found that the mutated CTTNBP2NL could not increase the phosphorylation level of STAT3 tyrosine 705 ( FIG. 3 ). Since serine is mutated to glutamic acid, phosphorylated serine can be imitated. Therefore, the phosphorylation of two serines at positions 488 and 527 can inhibit the phosphorylation effect of CTTNBP2NL on STAT3.

The experimental results show that: overexpression of CTTNBP2NL can cause increased phosphorylation of STAT3Y705 site, leading to disease; therefore, inhibiting the expression of CTTNBP2NL or inhibiting CTTNBP2NL to promote the phosphorylation activity of STAT3 protein in tumor cells can treat diseases caused by phosphorylation of STAT3 protein, especially Diseases caused by phosphorylation of STAT3 protein Y705. Phosphorylation of the 488th and 527th positions of CTTNBP2NL can effectively inhibit the phosphorylation of STAT3 by CTTNBP2NL.

Example 2. Knockout of CTTNBP2NL causes reduction of STAT3 phosphorylation in tumor cells

1. Experimental method

Lenti-CRISPRV2-CTTNBP2NL-guide RNA vector construction

The lentiCRISPR v2 vector was used as the eukaryotic expression vector, and the vector was purchased from Addgene. Use BsmBI of NEB Company for single enzyme digestion, enzyme digestion system: 50 μl, buffer: NEB Buffer 3.1, reaction time: 1h. Glue was recovered using the Tiangen Gel Recovery Kit.

The insert fragment is obtained by annealing into a double strand after synthesizing a single strand by the company. The annealing system: 20 μl, is carried out on a PCR machine, and the annealing conditions are: 95°C, 5min, 4°C, 5min.

The ligation was performed using T4 ligase from Quanjin Company, and the insert fragment and the digested lentiCRISPR v2 vector were ligated at a molar ratio of 7:1. Ligation conditions: overnight ligation at 16°C, and the ligation product was transformed into Stbl3 competent cells. The remaining steps are the same as the construction process of PLVX-CTTNBP2NL-3x Flag plasmid.

lentiviral packaging

The constructed viral backbone vector together with psPAX2 and pMD2.G packaging plasmids were transfected into 293T cells, lentiCRISPR v2:psPAX2:pMD2.G=1:2:1 (molar ratio). The cell supernatant was collected 48 hours after transfection, and the second cell supernatant was collected 72 hours after transfection. After concentrating the virus with PEG, aliquot it and temporarily store it in a -80°C refrigerator.

Stable knockout monoclonal cell line construction

293 cells were cultured in a 10cm cell culture dish and infected with CTTNBP2NL knockout lentivirus. After a period of infection, Puromycin was added to screen, and then the adherent cells were digested. After making a single cell suspension with DMEM medium containing penicillin and streptomycin, the Single cells are screened by flow cytometry. The obtained single cells continued to be cultured, and the growth status of the cells was observed under a microscope, and 20 monoclonal cells were selected for expansion culture, and the expression status of CTTNBP2NL was identified by Western-Blot method and gene sequencing method, and the CTTNBP2NL protein completely disappeared and the CTTNBP2NL CDS sequence was selected. Coded cells were used as a stable knockout monoclonal cell line.

Western blot

Use Co-IP and Western cell lysate from Beyontien Company. Specific steps: Take out the cultured cells or isolated mitochondria, wash the cell supernatant with PBS, add 100-400 μl of lysate according to the number of cells, and place on ice After 5 minutes, centrifuge at 12,000g at 4°C for 10 minutes, transfer the supernatant to a new EP tube, take out 5 μl for protein concentration determination, add 5X SDS protein sample buffer to the rest of the protein, place it on a constant temperature metal heat block, and denature at 95°C 5min, temporarily stored at -20°C.

Use the combination of 10% SDS separating gel + 5% SDS stacking gel for electrophoresis, electrophoresis conditions: 70V in the stacking gel, leave the stacking gel for about 20 minutes, and after entering the separating gel, adjust the voltage to 110V, and the separation time is about 60 minutes.

The membrane was transferred using SDS-methanol transfer buffer. Transfer conditions: 250mA, constant flow, transfer on ice for 90min, take out the PVDF membrane, block in 5% skimmed milk powder at room temperature for 30min, dilute the primary antibody with TBST, dilution ratio: 1:1000, 4°C, incubate overnight After the primary antibody was washed with TBST, the secondary antibody was added on a horizontal shaker at a dilution ratio of 1:2000, and incubated at 37°C for 1 hour. After washing the secondary antibody on a horizontal shaker with TBST, it was exposed on a chemiluminescence instrument and photographed.

2. Experimental results

STAT3 is an important member of the JAK-STAT family, which regulates important processes such as cell proliferation and apoptosis. In tumor cells, STAT3 is often hyperphosphorylated, leading to increased cell malignancy. Therefore, by knocking out the CTTNBP2NL gene, STAT3 hyperphosphorylation can be inhibited, thereby achieving the purpose of inhibiting tumor growth and treating tumors. In the present invention, CTTNBP2NL knockout cells were successfully constructed by CRISPR-Cas9 technology, and changes in STAT3 phosphorylation levels were detected by Western blot experiments. Using "ACTTTCATTGAAGAACGCTA", "GCCGCTGCCTTTCTTCCTCA", "TGTCACCTACATGCTAGAGA" as guideRNA targeting CTTNBP2NL, construct the above sequence into LentiCRISPR-GFP vector, infect Hela and HEK293 cells and select monoclonal cells, and obtain stable knockout cells by Western blot identification Tie. The results showed that in normal cells, STAT3 was not phosphorylated, and knocking out CTTNBP2NL in normal cells did not affect the phosphorylation status of STAT3. In tumor cells, STAT3 is always in a phosphorylated state. After CTTNBP2NL is knocked out, the phosphorylation of Y705 site of STAT3 is significantly reduced, while the phosphorylation of S727 site is not affected. The above results indicated that there was a correlation between the content of CTTNBP2NL and the content of phosphorylated STAT3, and after the content of CTTNBP2NL decreased, the content of phosphorylated STAT3 decreased accordingly ( FIG. 4 ).

Nucleotide sequence:

SEQ ID NO.5: ACTTTCATTGAAGAACGCTA

SEQ ID NO.6: GCCGCTGCCTTTCTTCCTCA

SEQ ID NO.7: TGTCACCTACATGCTAGAGA

The experimental results show that CTTNBP2NL gene can be knocked out by guideRNA targeting CTTNBP2NL, which can inhibit the expression of CTTNBP2NL, reduce the phosphorylation of Y705 site of STAT3, and treat diseases caused by phosphorylation of Y705 site of STAT3 protein.

Example 3. Knocking out or knocking down the expression of CTTNBP2NL in tumor cells leads to decreased cell proliferation and increased apoptosis of tumor cells

1. Experimental method

1. Interfering with lentiviral vector construction

(1) Synthesis of DNA fragments, annealing conditions: add 10 μl of sense strand + 10 μl of antisense strand DNA to a PCR tube, mix well, and place in a PCR instrument at 95°C for 5 minutes, 4°C for 5 minutes.

The DNA sequence is:

guide RNA: ACTTTCATTGAAGAACGCTA:

Upstream: CACCG ACTTTCATTGAAGAACGCTA (SEQ ID NO.8)

Downstream: AAACTAGCGTTTCTTCAATGAAAGTC (SEQ ID NO.9)

guide RNA: GCCGCTGCCTTTCTTCCTCA:

Upstream: CACCG GCCGCTGCCTTTCTTCCTCA (SEQ ID NO. 10)

Downstream: AAACTGAGGAAGAAAGGCAGCGGCC (SEQ ID NO. 11)

guide RNA: TGTCACCTACATGCTAGAGA:

Upstream: CACCG TGTCACCTACATGCTAGAGA (SEQ ID NO.12)

Downstream: AAACTCTCTAGCATGTAGGTGACAC (SEQ ID NO. 13)

(2) Double digestion of Plko.1 vector: AgeI+EcoRI.

(3) Ligate according to the ratio of carrier:fragment=1:7, 16°C, 12h.

(4) Transformation competence, monoclonal selection and sequencing, using U6 (sequence: ATGGACTATCATATGCTTACCGTA (SEQ ID NO.14)) as the sequencing primer, and forward sequencing.

2. Apoptosis detection

The Annexin V-PE/7-AAD cell apoptosis detection kit (product number: 40310ES20) of Shanghai Yisheng Biotechnology Co., Ltd. was used for detection, and the specific operation was carried out according to the manufacturer's instructions.

3. Cell proliferation detection

BeyoClick ^TM EdU-555 Cell Proliferation Detection Kit (product number: C0075S) of Beyotime Company was used for detection, and the specific operation was carried out according to the manufacturer's instructions.

2. Experimental results

In cultured HeLa cells in vitro, a knockout lentivirus expressing CTTNBP2NL caused decreased STAT3 phosphorylation, decreased cell proliferation, and increased apoptosis. Using "ACTTTCATTGAAGAACGCTA" (named guide1), "GCCGCTGCCTTTCTTCCTCA" (named guide2), "TGTCACCTACATGCTAGAGA" (named guide3) as guideRNAs targeting CTTNBP2NL, the proliferation was detected by the EdU insertion method, and the Annexin V-PE/7- AAD Cell Apoptosis Detection Kit was used to detect cell apoptosis. The results showed that knockdown of CTTNBP2NL significantly increased cell apoptosis (Fig. 5a), at the same time, cell proliferation was found to be significantly reduced (Fig. 5b).

In order to further determine the effect of CTTNBP2NL on tumor cell growth, we synthesized interfering RNA targeting the expression of CTTNBP2NL. The interfering sequences were: "ATAACCCAGTTAGGTATTATA (named sh-1)", "GGTACTCACTAAGCGTTTATT (named sh-2)", "CTGAACTCCTGACACTATTTA (named sh-3)", "GCCCTCCATCCAGGGATTTAT (named sh-4)", "GCCACTCCTGCTTACTCATAT (named sh-5)"; using liposome 3000 transfection, the above RNA fragments were transfected into Hela cells, and Observe the changes of cell proliferation and apoptosis. The results showed that interfering with the expression of CTTNBP2NL significantly reduced the cell proliferation ratio of Hela cells (Fig. 6a), and at the same time, cell apoptosis was found to be significantly increased (Fig. 6b).

Nucleotide sequence:

SEQ ID NO.15: ATAACCCAGTTAGGTATTATA

SEQ ID NO. 16: GGTACTCACTAAGCGTTTATT

SEQ ID NO.17: CTGAACTCCTGACACTATTTA

SEQ ID NO.18: GCCCTCCATCCAGGGATTTAT

SEQ ID NO.19: GCCACTCCTGCTTACTCATAT

In order to determine the effect of CTTNBP2NL knockout on the growth of tumor cells in vivo, CTTNBP2NL knockout cells were subcutaneously injected into nude mice, and the tumor growth was observed and recorded. The results showed that after knocking out CTTNBP2NL, tumor growth was significantly inhibited (Fig. 7).

In order to further determine the effect of CTTNBP2NL on the growth of tumor cells, an interference lentiviral vector targeting the expression of CTTNBP2NL was synthesized. The interference sequences were: "ATAACCCAGTTAGGTATTATA", "GGTACTCACTAAGCGTTTATT", "CTGAACTCCTGACACTATTTA", "GCCCTCCATCCAGGGATTTAT", "GCCACTCCTGCTTACTCATAT"; Cells were screened for stable knockdown of CTTNBP2NL monoclonal cells. CTTNBP2NL knockdown cells were subcutaneously injected into nude mice, and the tumor growth was observed and recorded. The results showed that knockdown of CTTNBP2NL significantly inhibited tumor growth (Figure 8).

The experimental results show that the use of interfering RNA targeting CTTNBP2NL expression can interfere with and reduce the expression of CTTNBP2NL gene, and significantly inhibit tumor growth.

Example 4, AAV-sh-CTTNBP2NL in the treatment of systemic lupus erythematosus

Systemic Lupus Erythematosus (SLE) is an autoimmune inflammatory connective tissue disease that mostly occurs in young women and often involves multiple organs. SLE is wreaking havoc on health and has many adverse effects on patients. The hyperphosphorylation of STAT3 plays an important role in SLE patients. Therefore, blocking the hyperphosphorylation of STAT3 by knocking down CTTNBP2NL can achieve the purpose of treating SLE. The present invention plans to construct and target CTTNBP2NL to interfere with AAV virus, and treat SLE through intravenous injection.

1. Experimental method

1. Interference with AAV virus construction:

(1) Synthesize DNA fragments, annealing conditions: add 10 ul sense strand + 10 antisense strand DNA to a PCR tube, mix well, and place in a PCR instrument at 95°C for 5 minutes and 4°C for 5 minutes.

The DNA sequence is:

Nucleotide sequence SEQ ID NO.15:

upstream:

GATCCATAACCCAGTTAGGTATTATACTCGAGTATAATACCTAACTGGGTTATTTTTTA (SEQ ID NO. 20)

Downstream:

AGCTTAAAAAATAACCCAGTTAGGTATTATACTCGAGTATAATACCTAACTGGGTTATG (SEQ ID NO. 21)

Nucleotide sequence SEQ ID NO.16:

upstream:

GATCCGGTACTCACTAAGCGTTTATTCTCGAGAATAAACGCTTAGTGAGTACCTTTTTA (SEQ ID NO. 22)

Downstream:

AGCTTAAAAAGGTACTCACTAAGCGTTTATTCTCGAGAATAAACGCTTAGTGAGTACCG (SEQ ID NO. 23)

Nucleotide sequence SEQ ID NO.17:

upstream:

GATCCCTGAACTCCTGACACTATTTACTCGAGTAAATAGTGTCAGGAGTTCAGTTTTTA (SEQ ID NO. 24)

Downstream:

AGCTTAAAAACTGAACTCCTGACACTATTTACTCGAGTAAATAGTGTCAGGAGTTCAGG (SEQ ID NO. 25)

Nucleotide sequence SEQ ID NO.18:

upstream:

GATCCGCCCTCCATCCAGGGATTTATCTCGAGATAAATCCCTGGATGGAGGGCTTTTTA (SEQ ID NO. 26)

Downstream:

AGCTTAAAAAGCCCTCCATCCAGGGATTTATCTCGAGATAAATCCCTGGATGGAGGGCG (SEQ ID NO. 27)

Nucleotide sequence SEQ ID NO.19:

upstream:

GATCCGCCACTCCTGCTTACTCATATCTCGAGATATGAGTAAGCAGGAGTGGCTTTTTA (SEQ ID NO. 28)

Downstream:

AGCTTAAAAAGCCACTCCTGCTTACTCATATCTCGAGATATGAGTAAGCAGGAGTGGCG (SEQ ID NO. 29)

(2) Double digestion of pAAV-ZsGreen-shRNA vector: BamHI+HindIII.

(3) Ligate according to the ratio of carrier:fragment=1:7, 16°C, 12h.

(4) Transformation competence, single clone selection and sequencing, using U6 as sequencing primer, forward sequencing.

2. Interfering with AAV virus treatment:

(1) The humanized immune system mice successfully induced SLE were taken and injected with AAV virus after anesthesia.

(2) Virus dosage: 1×10 ¹¹ GC/mouse, diluted to 100 μl, and injected into the tail vein with a microinjector.

(3) After 15 days of AAV virus injection, the phenotype changes of the mice were observed.

2. Experimental results

Using female adult MRL/lpr systemic lupus erythematosus (SLE) mice, an interfering RNA-expressing AAV virus of CTTNBP2NL was injected through the tail vein, successfully alleviating symptoms. By weighing body weight, it was found that knocking down CTTNBP2NL with AAV virus could significantly reduce the weight loss trend of SLE mice (Figure 9), and it was also found that knocking down CTTNBP2NL could significantly reduce the production of autoantibodies in SLE model mice (Figure 10).

The experimental results show that: inhibiting the expression of CTTNBP2NL can treat systemic lupus erythematosus.

Example 5, AAV-sh-CTTNBP2NL in the treatment of rheumatoid arthritis

Rheumatic arthritis (RA) is an autoimmune and inflammatory disease that usually results when the immune system mistakenly attacks the patient's own healthy cells, causing inflammation (painful swelling) in the affected body part. RA primarily attacks joint tissue, often multiple joints at a time, and the joints of the hands, wrists, and knees are all affected. Studies have found that diseased immune cells in RA patients often exhibit hyperphosphorylation of STAT3. Therefore, blocking the hyperphosphorylation of STAT3 by knocking down CTTNBP2NL can achieve the purpose of treating RA. The present invention plans to construct and target CTTNBP2NL to interfere with AAV virus, and treat RA through intravenous injection.

The method for constructing the interference AAV virus targeting CTTNBP2NL is the same as that in Example 4.

1. Experimental method

(1) Mix bovine type II collagen and Freund's complete adjuvant in a volume ratio of 1:1;

(2) Take 50 female adult-derived immune system DBA/1J mice, and inject 0.1mg subcutaneously at the base of the tail of each mouse

(3) The second injection after 25 days;

(4) After 50 days, the affected mice were treated with AAV: virus dosage: 1×10 ¹¹ GC/mouse, diluted to 100 μl, and injected into the tail vein with a microinjector.

(5) After 15 days of AAV virus injection, the phenotype changes of the mice were observed.

2. Experimental results

Rheumatoid arthritis was induced in female adult DBA/1J mice, and the interfering RNA expressing AAV virus of CTTNBP2NL was injected through the tail vein, and the symptoms were successfully relieved. By detecting the swelling thickness of the paw, it was found that knocking down CTTNBP2NL with AAV virus could significantly reduce the symptoms of rheumatoid arthritis mice (Figure 11).

The experimental results show that: inhibiting the expression of CTTNBP2NL can treat rheumatoid arthritis.

Example 6, AAV-sh-CTTNBP2NL in the treatment of inflammatory bowel disease

Inflammatory bowel disease (IBD) is a type of autoimmune disease that causes intestinal inflammation. IBD usually leads to persistent inflammation of the colon and rectum (large intestine or large intestine), causing bloody diarrhea in patients. The study found that diseased immune cells in IBD patients often exhibit hyperphosphorylation of STAT3. Therefore, blocking the hyperphosphorylation of STAT3 by knocking down CTTNBP2NL can achieve the purpose of treating IBD. The present invention plans to treat IBD through intravenous injection by constructing a CTTNBP2NL-targeted interference AAV virus.

The method for constructing the interference AAV virus targeting CTTNBP2NL is the same as that in Example 4.

1. Experimental method

(1) Dissolving dextran sodium sulfate (DSS) with water into a 5% solution;

(2) The humanized immune system C57BL/6 mice were repeatedly stimulated by drinking water administration, with an interval of one week and a total of 2 months of stimulation;

(3) AAV treatment was performed on the diseased mice: virus dosage: 1×10 ¹¹ GC/mouse, diluted to 100 μl, and injected into the tail vein with a microinjector.

(4) After 15 days of AAV virus injection, the phenotype changes of the mice were observed.

2. Experimental results

Female adult C57BL/6 mice with humanized immune system were purchased, fed for one week in an SPF environment, and a mouse model of inflammatory bowel disease was established by oral DSS. Mice with inflammatory bowel disease were treated by tail vein injection of CTTNBP2NL interfering RNA-expressing AAV virus. By detecting the content of inflammatory factors in intestinal tissue, it was found that knocking down CTTNBP2NL with AAV virus could significantly reduce the symptoms of inflammatory bowel disease mice (Figure 12).

The experimental results showed that inhibiting the expression of CTTNBP2NL could treat inflammatory bowel disease.

Example 7, AAV-sh-CTTNBP2NL in the treatment of psoriasis

Psoriasis is an autoimmune disease that usually manifests as skin disease, but it can also cause damage to various organs. Psoriasis usually causes red, itchy scales on the skin, most commonly on the knees, elbows, trunk, and scalp. Psoriasis is a common, chronic disease that is still completely cured. The onset of psoriasis has a certain periodicity, and the disease usually subsides for a period of time or enters a remission period after several weeks or months of onset. But continue to enter the onset period after a period of time. The study found that diseased immune cells in patients with psoriasis often exhibit hyperphosphorylation of STAT3. Therefore, blocking the hyperphosphorylation of STAT3 by knocking down CTTNBP2NL can achieve the purpose of treating psoriasis. The present invention plans to construct and target CTTNBP2NL to interfere with AAV virus, and treat psoriasis through intravenous injection.

The method for constructing the interference AAV virus targeting CTTNBP2NL is the same as that in Example 4.

1. Experimental method

(1) About 7-8 weeks old female humanized immune system Bab/c mice were treated with shaving and depilatory cream to expose the back skin.

(2) Apply 5% imiquimod cream (dose: 50 mg/cm ² ) to the depilated area, once a day for 15 consecutive days.

(3) Intraperitoneal injection of IFN-a (dose: 20 U/g body weight), administered once a day for 15 consecutive days.

(4) After the appearance of skin lesions, the affected mice were treated with AAV: virus dosage: 1×10 ¹¹ GC/mouse, diluted to 100 μl, and injected into the tail vein with a microinjector.

(5) After 15 days of AAV virus injection, the mice were sacrificed, the back skin was cut off to make a single cell suspension, and the changes in the proportion of immune cell subsets were determined by staining and flow cytometry analysis.

2. Experimental results

SPF grade humanized immune system BALB/c mice were bred for 7 weeks, and female mice were selected for psoriasis induction. Psoriasis model mice were treated by tail vein injection of CTTNBP2NL interfering RNA-expressing AAV virus. By detecting the changes in the proportion of immune cells in psoriasis lesions in mice, it was found that knocking down CTTNBP2NL with AAV virus could significantly reduce the symptoms of psoriasis mice (Figure 13).

The experimental results show that: inhibiting the expression of CTTNBP2NL can treat psoriasis.

Example 8. Effect of Overexpression of CTTNBP2NL on Tumor Cell CTTNBP2NL Phosphorylation

In order to determine the function of serine 568 of CTTNBP2NL protein, lentiviral vectors expressing CTTNBP2NL protein S568A (an inactivating mutation that cannot be phosphorylated) and S568E (an activating mutation that cannot be phosphorylated) were constructed, and infected Hela cells, and injected into Hela Cells were transfected with a dual-luciferase reporter plasmid for transcriptional activity downstream of STAT3. The effect of overexpression of different CTTNBP2NL mutants on STAT3 transcriptional activity can be judged by the ratio between firefly luciferase and Renilla luciferase. The results showed ( FIG. 14 ), the inactivating mutation at the S568 site overexpressing CTTNBP2NL did not affect the transcriptional activity of STAT3, but the activating mutation at the S568 site overexpressing CTTNBP2NL significantly inhibited the transcriptional activity of STAT3. The above results suggest that the S568 site is also a key site for the function of CTTNBP2NL.

In conclusion, the overexpression of CTTNBP2NL will increase the phosphorylation of STAT3Y705 site, leading to the occurrence of disease. Losing the function of CTTNBP2NL, such as inhibiting the expression of CTTNBP2NL or inhibiting CTTNBP2NL to promote the phosphorylation activity of STAT3 protein in tumor cells, can treat diseases caused by phosphorylation of STAT3 protein, especially the disease caused by phosphorylation of Y705 site of STAT3 protein. The loss of CTTNBP2NL function can effectively reduce the phosphorylation of the Y705 site of STAT3 protein in tumor cells, significantly increase tumor cell apoptosis, inhibit tumor cell proliferation, and then inhibit tumor growth; at the same time, the loss of CTTNBP2NL function can reduce the production of autoantibodies, and the therapeutic system lupus erythematosus; it can also reduce the symptoms of rheumatoid arthritis, inflammatory bowel disease and psoriasis, and effectively treat rheumatoid arthritis, inflammatory bowel disease and psoriasis. Therefore, the reagent for loss of CTTNBP2NL function can be used as a drug for treating tumors, autoimmune diseases and neurological diseases, and has a good application prospect.

SEQUENCE LISTING

<110> West China Hospital of Sichuan University

<120> Use of a Reagent for Deleting CTTNBP2NL Function in the Preparation of Drugs for Treating Diseases

<130> GYKH1669-2020P0111236CC

<160> 29

<170> PatentIn version 3.5

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Claims (3)

1. The use of a reagent for CTTNBP2NL function loss in the preparation of medicines for treating diseases caused by phosphorylation of STAT3 proteins, characterized in that: the diseases caused by phosphorylation of STAT3 proteins are cervical cancer, rheumatoid arthritis, systemic Lupus erythematosus, inflammatory bowel disease, psoriasis; the reagent that causes CTTNBP2NL function loss is a reagent that inhibits the expression of CTTNBP2NL or a reagent that inhibits CTTNBP2NL and promotes the phosphorylation activity of STAT3 protein in tumor cells; the reagent that inhibits the expression of CTTNBP2NL is RNAi , CRISPR/Cas9 method reagent; Said RNAi method uses the reagent to target the RNA sequence of CTTNBP2NL as: ATAACCCAGTTAGGTATTATA, GGTACTCACTAAGCGTTTATT, CTGAACTCCTGACACTATTTA, GCCCTCCATCCAGGGATTTAT, GCCACTCCTGCTTACTCATAT; The RNA sequence targeting CTTNBP2NL of reagents for the CRISPR/Cas9 method is: ACTTTCATTGAAGAACGCTA, GCCGCTGCCTTTCTTCCTCA, TGTCACCTACATGCTAGAGA; The reagent for inhibiting CTTNBP2NL from promoting STAT3 protein phosphorylation activity in tumor cells is a reagent that mutates the 488th serine of CTTNBP2NL to glutamic acid and/or the 527th serine to glutamic acid. 2. The use according to claim 1, characterized in that: the drug is a drug for treating diseases caused by phosphorylation of Y705 site of STAT3 protein. 3. The use according to claim 1 or 2, characterized in that: the reagent for inactivating CTTNBP2NL function is carried by adenovirus, adeno-associated virus, lentivirus or cells.

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